Cancer Research Institute, Xiang-Ya School of Medicine, Key Laboratory for Carcinogenesis of Chinese Ministry of Health, Key Laboratory for Carcinogenesis & Cancer Invasion of Chinese Ministry of Education, Central South University, Xiangya Road 110, 410078, Changsha, Hunan, P R China.
Mol Cancer. 2013 Jun 8;12:53. doi: 10.1186/1476-4598-12-53.
Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.
Immunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).
The positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.
Our study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.
鼻咽癌（NPC）是中国南方和东南亚地区常见的恶性肿瘤，但发病机制的分子机制仍不清楚。我们之前的工作表明，BCAT1 mRNA 在 NPC 中过表达，在 5-8F NPC 细胞系中敲低其表达能强烈抑制细胞周期进程和细胞增殖。然而，BCAT1 上调的机制及其在 NPC 发展中的功能作用仍有待阐明。
采用免疫组织化学（IHC）方法检测不同病理阶段 NPC 中 BCAT1 蛋白的表达。利用 PCR 测序、定量聚合酶链反应（qPCR）、IHC、染色质免疫沉淀（ChIP）和荧光素酶报告系统分别分析基因突变、DNA 扩增和转录因子 c-Myc 对 BCAT1 表达的调控作用。通过 RNA 干扰（RNAi）评估 BCAT1 在集落形成、细胞迁移和侵袭特性中的功能。
正常上皮、低-中级别异型增生组织、高级别异型增生组织和 NPC 组织中 BCAT1 蛋白表达的阳性率分别为 23.6%（17/72）、75%（18/24）、88.9%（8/9）和 88.8%（71/80）。仅在外显子 1 中检测到一个 SNP 位点，42.4%（12/28）的 NPC 组织显示 BCAT1 微卫星位点扩增。c-Myc 可直接结合 BCAT1 启动子区域的 c-Myc 结合位点，上调其表达。53.6%（15/28）和 59.1%（13/22）的 NPC 组织中同时表达 c-Myc 和 BCAT1 的 mRNA 和蛋白，c-Myc 敲低细胞系中 BCAT1 mRNA 表达也下调。此外，BCAT1 敲低细胞表现出增殖能力降低、细胞迁移和侵袭能力降低。
本研究表明，基因扩增和 c-Myc 上调是原发性 NPC 中 BCAT1 过表达的原因，BCAT1 过表达诱导细胞增殖、迁移和侵袭。结果表明，BCAT1 可能是 NPC 诊断和治疗的新分子靶点。
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